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Armelle Corpet, Constance Kleijwegt, Simon Roubille, Franceline Juillard,

Karine Jacquet, Pascale Texier, Patrick Lomonte

To cite this version:

Armelle Corpet, Constance Kleijwegt, Simon Roubille, Franceline Juillard, Karine Jacquet, et al..

PML nuclear bodies and chromatin dynamics: catch me if you can!. Nucleic Acids Research, Oxford

University Press, 2020, 48 (21), pp.11890-11912. �10.1093/nar/gkaa828�. �hal-03043659�

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SURVEY AND SUMMARY

PML nuclear bodies and chromatin dynamics: catch

me if you can!

Armelle Corpet

*

, Constance Kleijwegt, Simon Roubille, Franceline Juillard,

Karine Jacquet, Pascale Texier and Patrick Lomonte

*

Univ Lyon, Universit ´e Claude Bernard Lyon 1, CNRS UMR 5310, INSERM U 1217, LabEx DEVweCAN, Institut NeuroMyoG `ene (INMG), team Chromatin Dynamics, Nuclear Domains, Virus F-69008, Lyon, France

Received June 11, 2020; Revised September 15, 2020; Editorial Decision September 16, 2020; Accepted September 18, 2020

ABSTRACT

Eukaryotic cells compartmentalize their internal mi-lieu in order to achieve specific reactions in time and space. This organization in distinct compart-ments is essential to allow subcellular processing of regulatory signals and generate specific cellu-lar responses. In the nucleus, genetic information is packaged in the form of chromatin, an organized and repeated nucleoprotein structure that is a source of epigenetic information. In addition, cells orga-nize the distribution of macromolecules via various membrane-less nuclear organelles, which have gath-ered considerable attention in the last few years. The macromolecular multiprotein complexes known as Promyelocytic Leukemia Nuclear Bodies (PML NBs) are an archetype for nuclear membrane-less or-ganelles. Chromatin interactions with nuclear bodies are important to regulate genome function. In this re-view, we will focus on the dynamic interplay between PML NBs and chromatin. We report how the struc-ture and formation of PML NBs, which may involve phase separation mechanisms, might impact their functions in the regulation of chromatin dynamics. In particular, we will discuss how PML NBs participate in the chromatinization of viral genomes, as well as in the control of specific cellular chromatin assembly pathways which govern physiological mechanisms such as senescence or telomere maintenance. INTRODUCTION

Eukaryotic cells package ∼2 m of DNA into a nucleus of a few micrometers diameter together with all the

bio-logical macromolecules required to organize, replicate, and interpret this genetic information. Mechanisms have thus evolved to organize this crowded environment. Our genetic material is packaged in a complex nucleoprotein structure called chromatin, whose basic unit, the nucleosome is com-posed of an octamer of histones comprising two copies of each core histone H2A, H2B, H3 and H4, around which is wrapped 147 bp of DNA (1). Targeted deposition of histone variants, or addition of specific post-translational modifi-cations to histones and DNA provide a large repertoire of epigenetic information that can modulate chromatin acces-sibility and gene expression, and thus regulate cell iden-tity (2). On the other hand, spatial and temporal distribu-tion of macromolecules is organized through membrane-bound and membrane-less organelles which participate in the compartmentalization of biochemical reactions in the nucleus. Liquid–liquid phase separation (LLPS) has re-cently emerged as a new biophysical paradigm providing a mechanistical basis for membrane-less organelles assem-bly in a spontaneous manner (3–8). Upon specific bio-physical conditions (pH, temperature, concentration, na-ture of the macromolecule, etc.), biological macromolecules concentrate in phase-separated liquid-like droplets, which coexist with a dilute phase, like oil drops in water. This process is energetically favorable and allows the formation of membrane-less compartments, called biomolecular con-densates (3,4).

Promyelocytic leukemia (PML) nuclear bodies (NBs) (also known as ND10) are an archetype of membrane-less organelles, that concentrate proteins at discrete sites within the nucleoplasm (9,10). They form a sphere of∼0.1–1 ␮m in diameter and are present in the majority of mammalian cell nuclei (9). PML NBs were discovered through their disorga-nization in acute promyelocytic leukemia (APL). The PML gene was identified at the breakpoint of a common

translo-*To whom correspondence should be addressed. Tel: +33 4 26 68 82 58; Fax: +33 4 26 68 82 92; Email: armelle.corpet@univ-lyon1.fr

Correspondence may also be addressed to Patrick Lomonte. Tel: +33 4 26 68 82 57; Fax: +33 4 26 68 82 92; Email: patrick.lomonte@univ-lyon1.fr

C

The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License

(http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work

is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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cation t(15,17) resulting in a fusion protein with retinoic acid receptor alpha (RAR␣) that drives APL (11). The tumor-suppressor PML protein is the main organizer of PML NBs and forms a shell surrounding an inner core of dozens of proteins (12). PML NBs have been implicated in a wide range of biological processes such as senescence, an-tiviral defense or stemness. They may act both to concen-trate components to facilitate biochemical reactions such as sumoylation or as storage compartments regulating protein availability in the nucleus.

In this review, we will discuss how the biophysical pro-cess of LLPS may participate in the multi-step biogenesis of PML NBs (3,6). We will consider the interplay between PML NBs and the regulation of chromatin dynamics in light of this new paradigm of phase separation and explore its various functional implications. We will provide some perspective on how the partitioning of various chromatin-related factors in the PML NBs might provide a means to fine-tune gene expression and chromatin plasticity.

STRUCTURE AND FORMATION OF PML NBs Structure of PML and PML NBs

The structure of PML NBs has been extensively studied using both light and electron microscopy. There are typi-cally 5–30 PML nuclear bodies per nucleus depending on the cell type, cell-cycle phase or physiological state (13). In immunofluorescence, they appear as nuclear dot-shaped spherical structures that reside in the interchromatin nu-clear space (14). By electron or super-resolution light mi-croscopy, it was observed that PML protein is concentrated in a≈100 nm thick shell at the periphery of nuclear bod-ies, surrounding an inner core filled with dozens of fac-tors (15,16). More than 170 proteins have been found to re-side either constitutively or transiently in PML NBs (12). Among them, the nuclear antigen Sp100 was the first char-acterized protein to localize in these nuclear bodies (17) and is found together with PML in the periphery (16).

The PML protein (also known as TRIM19) is an es-sential component of PML NBs (Figure 1A). PML be-longs to the family of tripartite motif (TRIM)-containing proteins characterized by a conserved RBCC motif con-sisting of a RING finger domain (R) followed by two cysteine-histidine-rich B-box domains (B) and an alpha-helical coiled-coil domain (CC). While alternative splicing of C-terminal exons generates seven different isoforms of PML, they all contain the conserved RBCC motif in their N-terminal part (13). All PML isoforms, except PML-VII, show a predominantly nuclear pattern due to the conser-vation of the nuclear localization signal (NLS) present in exon 6, but may also display specific functions that will not be discussed in the present study (for review (18)). PML protein sustains multiple post-translational modifica-tions including SUMOylation, the covalent attachment of a small ubiquitin-like modifier (SUMO) protein to a protein-specific lysine residue (19). PML main SUMOylation sites are lysines K65, K160 and K490 (20), although SUMOyla-tion of other lysines, like K616 has also been reported (21). PML also contains a SUMO interacting motif (SIM) at po-sition aa 556–562 enabling it to interact with SUMOylated proteins (22) (Figure1A). PML’s branched SUMO chains

and SIM motifs may provide a ‘molecular glue’ to stabilize proteins within PML NBs (see below).

Formation of PML NBs and LLPS

While early models put forward an interaction between PML-conjugated SUMO and PML-SIM to nucleate NBs (22–24), recent analyses brought new insights on the multi-step formation of PML NBs and the recruitment of their protein constituents possibly through phase separation mechanisms.

PML, which is the main organizer of PML NBs, is a mul-tivalent protein with multiple modular domains and inter-action motifs, a key feature that can enable polymerization-driven liquid-liquid phase separation. PML is essential for the structural integrity of PML NBs (25) and is therefore referred to as a scaffold protein. Other proteins that per-manently or transiently reside in PML NBs are called client proteins (6). The first phase of PML NB formation relies on intermolecular covalent disulfide linkage of oxidized PML monomers, as well as non-covalent interactions between the RBCC domains to drive assembly of PML multimers form-ing the primary nuclear body outer shell (24,26,27). These PML multimers are absent in MEF Pml–/– cells reconsti-tuted with a PML mutated in the RBCC domain, empha-sizing the importance of this domain for nuclear body for-mation (27). Recent crystallographic studies of PML RING and B1 domains put forward a cooperative mechanism in which a RING tetramerization step is followed by B1 poly-merization of PML to allow macromolecular scaffolding of PML (28,29). On the contrary, PML SUMO-mutants (e.g. PML 3KR), or devoid of their SIM (PMLSIM) al-low the formation of PML multimers and the formation of spherical PML NBs exactly like the WT structures when introduced into MEFs Pml–/– (27). This underscores the non-essential role of the PML SUMO-SIM interactions for the initial steps of PML NB formation. Of note, stud-ies also point out the importance of C-terminal regions of specific PML isoforms in the oligomerization process of PML (30,31). The next step involves the recruitment of UBC9, the only SUMO E2-conjugating enzyme known so far, which is dependent on the RBCC oligomerization of PML (28). UBC9-mediated SUMOylation of PML then regulates and enforces PML-PML interactions via inter-molecular SUMO-SIM interactions consistent with their importance to form mature PML NBs (22–24,27) (Figure 1B). In addition, SUMOylated PML drives the multiva-lent recruitement of inner core client proteins through their SIM, to form mature PML NBs (see below).

Membrane-less organelles biogenesis has recently been revisited through the prism of LLPS which stipulates that above a concentration threshold some proteins may phase separate and form liquid-like droplets with a distinct composition from the surrounding environment (3,8,32). Beautiful in vitro experiments demonstrated that mixes of polySUMO-polySIM polymers allow droplet forma-tion that can recruit SUMO/SIM clients depending on the number of free sites remaining on the polymer (6). When transfected into cells, these polymers trigger the formation of condensates, that can be induced specifically at telom-eres, and regulate partitioning of SUMO/SIM-containing

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A

R B1

RBCC motif

B2

Exons 1-6 Exons 7-9 : alternative splicing

CC B SIM C PML scaffold protein S S S client protein SIM S PML NB outer shell chromatin fiber inner core

List of chromatin-related factors ATRX CBP DAXX DEK H3.3 HDAC7 HIRA complex HP1 1. Oxidative stress 3. UBC9-mediated PML SUMOylation inner core (ii) (i) chromatin PML NB S S S S S S S S S S S S S SS S S S S 2. Disulfide bond formation and RBCC mediated oligomerization 6. In situ client SUMOylation : enforcement of client localization in PML NBs via SIM-SUMO interactions S S S S S S S S S S S S S S S S S S S S S SS S S S S S S S S S S S S S S S S S S S S PML NB outer shell S S S SS S S NLS UBC9 UBC9 SIM S SUMO1 S SUMO2/3 PML disulfide bond formation RBCC weak non-covalent interactions S 4. Self organization

5. Client recruitment via SIM interactions with SUMOylated PML MOZ SATB1 SETDB1 SIRT1 TET2 TIP60

Figure 1. Structure of PML and organization of PML NBs. (A) Structure of the PML protein scaffold. All PML isoforms (I–VII), ranging from 882aa (PML-I) to 435aa (PML-VII), possess a conserved RBCC/TRIM motif in their N-terminal part. The different C-terminal parts of PML-I to VI are generated through alternative splicing of the 3exons 7 to 9 of the unique PML gene, while PML-VII only possesses exons (1–4 and 7b). SUMO modification sites (S) are indicated at lysine positions K65, K160, K490 and K616. The NLS (Nuclear Localisation Sequence) and the SIM (SUMO Interacting Motif) are indicated. NB: PML structure is not to scale. (B) Formation of canonical PML NBs. (1) Oxidative stress triggers PML cross-linking by disulfide bond formation. (2) Together with RBCC weak non-covalent interactions, this triggers oligomerization of non SUMOylated PML proteins. (3) UBC9-mediated (poly-)SUMOylation of PML then allows multiple SUMO-SIM interactions, (4) which stabilize the formation of the self-organized matrix-associated outer shell, possibly involving liquid-liquid phase separation mechanisms. Of note SUMO1 modification (yellow) is mostly present in the PML NB outer shell, while the poly-SUMO2/3 chains (orange) present in the shell also protrude to variable degrees in the interior of the PML NB. (5) Client proteins are recruited in the outer shell (eg Sp100 not shown) as well as in the inner core through specific interactions of their SIM with the SUMOylated-PML scaffold. (6) UBC9 SUMOylation of client proteins then enforces their sequestration in PML NBs. Turnover of client proteins is relatively rapid ranging from seconds to a few minutes. (C) PML NBs are interspersed in the chromatin. (left) Immunofluorescence analysis of human primary BJ fibroblasts stained by PML (green) and DAPI (red). Scale bar is 10␮m. (right) Scheme showing PML NBs (green) surrounded by chromatin loops (red). Cellular loci, such as telomeres, can localize partly within PML NBs in specific cases (i) (see main text). Chromatin-related factors (histone modifiers, histone readers and histone chaperones) as well as viral genomes (ii) localize inside PML NBs.

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clients (6,33,34). While PML is a multivalent protein with several identified SUMO/SIM interaction modules that could contribute to a possible phase separation of PML NBs, phase transition properties of the PML protein itself have yet to be demonstrated. In addition, this SUMO/SIM condensation process is not sufficient alone to explain the specific architecture of PML NBs, which exhibit a spher-ical shell formed by the oligomerized PML protein sur-rounding an inner core of client proteins (35). This dual phase architecture is rather unique among membrane-less organelles, and it remains to be determined to what ex-tent the shell and the inner core present different solid-like versus liquid-like biophysical properties. Yet, the existence of multiphase biomolecular condensates, formed by LLPS such as the nucleolus (3,36) does not exclude the possible contribution of LLPS to PML NBs biogenesis. In particu-lar, one hallmark feature of the LLPS model, ie, the con-centration buffering/dependence, is validated by PML NBs (3,8,37). Size of the PML NBs scales up when increasing the concentration of PML as observed upon IFN-I treatment or senescence entry (38–42). On the contrary PML NBs are dissolved when artifically expanding the volume of the nu-cleus (43). In addition, PML NBs exhibit many other prop-erties that meet the criteria defining LLPS-based structures, including a spherical shape, fusion/fission events in physi-ological or stress conditions, or high molecular mobility of internal components (Table1). Nevertheless, many of this evidence remains qualitative and only provides indirect ev-idence for LLPS in vivo (32).

It is also important to discriminate between true LLPS and alternative mechanisms that could concentrate factors in a given place. In particular, it was recently shown that the transient non-specific binding of RNA polymerase II to the naked DNA of the herpes simplex virus 1 (HSV-1) genome during the lytic phase, leads to a viral DNA-mediated nu-clear compartmentalization of replication foci through a mechanism distinct from LLPS (44), and rather driven by polymer-polymer phase separation (PPPS) (for review (37)). This chromatin bridging mechanism also explains the for-mation of heterochromatin foci that behave as collapsed chromatin globules (45), despite the fact that Heterochro-matin Protein 1 (HP1) can undergo LLPS (46,47). As de-scribed above, several lines of evidence rather support a con-tribution of LLPS mechanisms for PML NBs biogenesis, in-dependently of the chromatin polymer (Table1). Yet, pres-ence of DNA nucleation sites may help to recruit and con-centrate PML proteins to reach the saturation concentra-tion required for PML NBs droplet formaconcentra-tion. PML NBs can be formed de novo at telomeric DNA and subsequently detach from them, suggesting that a nucleation site could mediate the formation of a subset of PML NBs (48). In ad-dition, forced tethering of PML proteins to chromatin by the LacO/LacI or dCas9 systems induces PML NB forma-tion at the targeted locus, suggesting that chromatin-bound PML proteins could be seeds for PML NB formation at spe-cific loci by reaching the saturation concentration for con-densate formation (45,49–51).

Many membrane-less organelles, such as nucleolus or Ca-jal bodies, are condensates that contain, in addition to pro-teins, RNA molecules which are critical for LLPS. PML NBs are found in regions of high transcriptional

activ-ity (14, 52) and early studies showed that nascent RNA could be found inside PML NBs in normal conditions (53) or upon interferon (IFN)␣ or IFN␥ stimulation (54,55). In addition, it was recently shown that the long non-coding RNA (lncRNA) telomeric repeat-containing RNA (TERRA) is found within PML NBs of cells that activate a specific telomere maintenance pathway (56) (see below). Yet, it remains controversial to what extent bulk RNAs physically localize within PML NBs. Other publications showed that nascent RNAs are not enriched in PML NBs (57) but rather accumulate in their vicinity together with highly acetylated blocks of chromatin (14). In addition, brief transcriptional inhibition does not dramatically im-pact PML NBs structure (54,58), in contrast to nucleoli (59,60), and RNAs are not required per se for PML NBs biogenesis (6).

PML NBs thus appear as membrane-less organelles formed through a multi-step process that initially involves PML polymerization-driven shell formation followed by multivalent SUMO–SIM interactions of the PML scaffold and partners that could regulate liquid–liquid phase separa-tion of PML NBs and their composisepara-tion (see below), with-out any contributing RNA. We refer hereafter to these nu-clear bodies as canonical PML NBs.

Alternative PML-containing structures

Remarkably, a continuum of PML-containing structures has been observed, in which the liquid-like properties of canonical PML NBs seem to be lost. After entry into mi-tosis, PML NBs undergo dramatic rearrangements (61), and partition in distinct larger aggregates of PML proteins called MAPPs (Mitotic Accumulation of PML Protein) (62) (for review (63)). MAPPs neither undergo rapid exchange of the PML protein, nor fusion/fission processes (62), that are essential criteria for LLPS (Table1). PML undergoes an extensive de-SUMOylation preventing it from recruiting SIM-containing proteins such as the regular components of PML NBs, Sp100 or Death Domain-Associated Pro-tein (DAXX) (61,62) (see below). In early G1, PML NBs, in the form of the so-called CyPNs (Cytoplasmic assem-blies of PML and Nucleoporins), become associated with karyopherin KPNB1 which recruits FG-repeat-containing nucleoporins. The latter have the ability to form an hydro-gel that encapsulates PML aggregates, which could facili-tate their re-solubilization and nuclear import (64,65). Se-quential recruitment of PML NBs components then oc-curs allowing reformation of mature canonical PML NBs (62,66). Other atypical PML-containing structures associ-ated with nuclear lipid droplets were observed in cells af-ter fatty acid stress (67,68). These so-called lipid-associated PML structures (LAPS) differ from canonical PML NBs as they lack SUMO1, Sp100 and DAXX proteins (68). PML only occupies part of the surface of the nuclear lipid droplets and is not required for their formation but is nec-essary for their functional maturation (68). In human em-bryonic stem cells (hESCs), PML-containing structures, also devoid of SUMO1, Sp100 or DAXX, show particu-lar morphological types in the forms of long-linear rods or rosettes (69), which may be used as an indicator of the pluripotent state of the cells (70). Finally, our data

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Table 1. Summary of LLPS criteria that are matched or not by canonical PML NBs. In this table, we put forward the experimental evidence that sustains or not the involvement of LLPS in biogenesis of canonical PML NBs. Criteria listed here have been chosen based on the following reviews (4,8,32) and may not be all necessary/sufficient to prove LLPS. n.d. : non determined

LLPS criterion

Criterion met by

PML NBs Experimental evidence References

Spherical shape (roundness): liquid droplets have a

spherical shape driven by surface tension

Yes Super-resolution microscopy or transmission electron microscopy of PML NBs show sphericity of these nuclear bodies

(15,16,27,35)

Fusion/fission: like oil droplets in water, biomolecular

condensates have the ability to fuse or drip

Yes Time-lapse observations of PML NBs confirms their ability to undergo fusion/fission events during DNA

replication or upon various stress conditions such as DNA damage, heat shock or physical pressure

(15,171–172)

Molecular mobilitya: liquid condensates are characterized by a high mobility of proteins within them which is essentially depending on diffusion

Partially FRAP experiments underlined fast recovery times for client proteins such as DAXX, CBP or BLM in the range of seconds, while PML isoforms exhibit slightly slower recovery times in the range of a few minutes compatible with the liquid-like nature of PML NBs. However, long recovery rates have been observed for specific isoforms such as PML V which may contribute to the structural integrity of nuclear bodies and could act as a stable scaffold for the recruitment of faster-exchanging molecules such as DAXX or CBP

(14–15,115)

Concentration buffering/dependence: LLPS is a function

of concentration: past the critical concentration required for droplet formation, production of more protein increases droplet size but does not change concentration in either phase

Yes Increase in PML intracellular concentration, as observed upon IFN-I treatment or senescence entry, results in an increased PML NBs size, while a decrease in PML protein concentration dissolves PML NBs in vivo

(38–43)

Interfacial boundaryb: phase-separated proteins should preferentially move within the droplet. Presence of a phase boundary should reduce diffusion across the boundary

Partially Diffusion coefficient for NLS-GFP was determined in nucleoplasm or in PML NBs by FCS. This demonstrated a 3-fold reduction in the diffusion coefficient inside the PML NBs as well as reduced exchanges of NLS-GFP between PML NBs and the nucleoplasm

(16,115)

Undergoes LLPS in vitro/in vivo Partially Not demonstrated for the PML protein itself. Yet, polySUMO-polySIM polymers form droplets in vitro and

in vivo and recruit SUMO/SIM containing protein clients in vitro and in vivo

(633–34)

Temperature/ion strenght/pH dependance: measure of

droplet formation in vivo should show dependance on temperature, ion concentration or pH

n.d. n.d.

-Sensitivity to 1,6-hexanediol: this chemical compound

perturbs weak hydrophobic interactions that are involved in LLPS. Yet, sensitivity to 1,6 hexanediol is neither necessary nor sufficient to demonstate that a structure is formed by LLPS

n.d. n.d.

-Optodroplet assay: investigate whether expression of a

fusion protein (protein of interest fused to a photolyase domain that can self-associate upon blue light) facilitates droplet formation in vivo upon blue light stimulation. Results should be interpreted with caution since these experiments rely on an artificial fusion protein system and should thus be combined with other experiments to prove LLPS in vivo

n.d. n.d.

-aMolecular mobility is traditionally measured by Fluorescence Recovery After Photobleaching (FRAP). However, it should be noted that the use of recovery time as a marker of LLPS is insufficient per se since rapid recovery can result from a variety of mechanisms (32). One critical point is to demonstrate that the recovery rate is truely dominated by diffusion (rather than binding), which can be assessed by performing FRAP with various sizes of the bleach spot (32), which has not been performed yet in PML NBs. bDiffusion across the boundary can be measured by fluorescence correlation spectroscopy (FCS) or single-molecule tracking (SMT). Alternatively, FRAP performed on half of the condensate, as performed in (45) provides an original and quantitative measure for the presence of a impermeable boundary, which could potentially be applied to PML NBs.

acterizing PML NBs in sensory neurons within human trigeminal ganglia show the presence of large aggregates of PML, lacking SUMO1, Sp100 and DAXX (71) (and unpublished). Thus, self-association of the PML protein scaffold allows to form various alternative PML-containing structures that do not exhibit LLPS properties. We hy-pothesize that the presence of SUMO1, which can undergo LLPS in vitro (6), is key to promote formation of canonical PML NBs possibly via triggering LLPS and thus

partition-ing of regular client proteins such as DAXX or Sp100 in PML NBs.

While canonical PML NBs appear as discrete foci inter-spersed between chromatin (Figure 1C), we will now fo-cus on understanding the physical and functional connec-tion of PML NBs with chromatin and investigate how the compartmentalization activity of PML NBs through phase separation mechanisms provides multiple strategies to reg-ulate chromatin-related factors partitioning and chromatin

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dynamics. In particular, we can envisage three main non-exclusive processes that will be discussed: (1) PML NBs may be hotspots for modifications such as SUMOylation, poten-tially modulating the activity of chromatin-related factors; (2) PML NBs may store/sequestrate chromatin-related fac-tors and control their dynamic release thereby fine-tuning the nucleoplasmic pool of a given factor and (3) PML NBs may help targeting chromatin-related factors to spe-cific chromatin-associated regions by compartmentalizing them (Figure2).

A CONNECTION OF PML NBS WITH CHROMATIN PML NBs contain multiple chromatin associated proteins, in-cluding histones and histone chaperones

The idea of a role for PML NBs in the regulation of chro-matin dynamics emerged via the identification of numerous chromatin-modifying factors within PML NBs, such as the CREB-Binding Protein (CBP), an histone acetyltransferase (HAT) involved in transcriptional regulation (53,72,73), or HP1 (74–77). HP1 is a key protein involved in heterochro-matin formation, which interacts with Sp100, a constitutive component of PML NBs, and which localizes within these bodies in interphase as well as in senescent cells (74–78), suggesting very early on a connection of PML NBs with chromatin dynamics. Together with HP1, DAXX was iden-tified as a constitutive PML NBs component (25,77) but it was not until its identification as a histone chaperone that the connection with chromatin dynamics was made (79). DAXX associates with the chromatin remodeler ATRX to form an H3.3-specific histone chaperone essential for H3.3 deposition at heterochromatin loci (79–81), and is required for ATRX localization in PML NBs (82–85) (Table2).

Histone chaperones are dedicated proteins, which as-sociate with non-nucleosomal histones and escort them throughout their cellular life in processes ranging from nuclear import, storage, assembly/disassembly onto chro-matin during several DNA metabolic processes (86). His-tone chaperones can be distinguished on the basis of their histone binding selectivity with a preference for H2A–H2B or H3–H4 histones and with additional selectivity towards specific histone variants. The replicative histone variants represent the bulk of histones and are expressed in S-phase, while replacement variants are expressed constitutively at lower levels. Among the H3-H4 histone chaperones, the CAF-1 complex is involved in the specific deposition of the H3.1 replicative histone variant in a DNA-synthesis dependent manner, while HIRA and DAXX-ATRX are H3.3 specific histone chaperones complexes implicated in H3.3–H4 deposition in a DNA-synthesis independent man-ner (for review (86)). H3.3 deposition was initially identi-fied as characteristic of euchromatic transcriptionally ac-tive regions with high histone turnover (87–89). HIRA in-teracts with RNA polymerase II (90), specific transcription factors (TFs) (91) or replication protein A (RPA) found in nucleosome-free regions (92), thus mediating H3.3 de-position at active regulatory elements such as enhancers, promoters or gene bodies (80,92). Although unexpected, H3.3 was later found enriched in heterochromatin loci such as telomeres or pericentromeric chromatin where it is de-posited by the DAXX-ATRX complex (79–81). This reflects

the double face of H3.3 histone variant in gene regulation, which is context-dependent (for review (86,93,94)).

Interestingly, PML NBs seem to have a strong connection with the H3.3 chromatin assembly pathway. In addition to the constitutive localization of the DAXX–ATRX complex in PML NBs, the HIRA H3.3-specific histone chaperone complex, composed of HIRA, UBN1, CABIN-1 and tran-siently ASF1A, also localizes in PML NBs upon senescence entry (78,95–97) and upon viral infection-associated type I interferon (IFN-I) signaling (98–100) (see below). Further-more, soluble newly-synthesized H3.3-H4 histone dimers are brought to PML NBs in a DAXX-dependent manner before deposition onto chromatin (84,85), thus suggesting that PML NBs may be important regulatory sites for the sorting of H3.3 among various histone chaperones com-plexes and further incorporation of H3.3 onto chromatin. Of note, another H3.3 specific chaperone called DEK also localizes in PML NBs in stem cells, and may participate in the maintenance of an H3.3 soluble pool available for asso-ciation with other chaperones in PML NBs (101) (Table2 and see below).

Finally, SETDB1, an histone H3 lysine 9 (H3K9) specific methyltransferase also localizes constitutively in PML NBs, which may be related to the transcriptional repression of specific genes (102). Given the connection of SETDB1 with DAXX–ATRX in the heterochromatinization of retroele-ments in mouse ES cells (103,104), further studies will be required to determine the function of the SETDB1 pool in PML NBs in regards to its function in heterochro-matin maintenance. Other H3K9 methyltransferases, such as SUV39H1 and G9a, or EZH2, an H3K27 methyltrans-ferase member of the Polycomb Repressive Complex 2 (PRC2), associate with PML but it remains to be shown whether they actually localize in PML NBs (105–107). In-terestingly, other histone modifiers, such as TIP60 (KAT5) and MOZ (KAT6A) histone acetyltransferases, histone deacetylase 7 (HDAC7), or SIRT1, partition in PML NBs to regulate chromatin dynamics and transcriptional regula-tion (108–112). Finally, PML NBs are also associated with DNA demethylation activities through the recruitment of the ten-eleven translocation dioxygenase 2 enzyme (TET2) in response to chemotherapy and exclusion of DNA methyl-transferase 3A (DNMT3A) (113,114) (Table2).

Regulation of the composition of PML NBs in chromatin-related factors

How do cells regulate the composition of PML NBs and what molecular mechanisms govern the recruitment of the chromatin-associated factors in PML NBs? Indeed, while PML NBs are macroscopically stable and persist for hours/days, photobleaching recovery experiments showed that they are highly dynamic at the molecular level, turning over their contents on time-scales from seconds to minutes (15,115). In light of the LLPS paradigm, we will now ex-plore how the partitioning of various histone chaperones in the membrane-less PML NBs is regulated. The number of interaction modules (valency) and their affinity are key pa-rameters controlling phase separation and could thus en-able compositional control of PML NBs (6). In particu-lar, changes in concentration or specific post-translational

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(1) concentration of biochemical reactions

(2) Buffering/sequestration of chromatin-related factors (3) Organization of specific chromatin domains (1)

S

client

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Three possible functions of PML NBs in relation to their liquid-like properties PML NB outer shell (3) UBC9 H3K4me3 SIM S SUMO1 S SUMO2/3 Nucleosome (H3-H4)*2 in gray + 2 H2A-H2B dimers in blue wrapped with 147bp DNA

active transcription

Modification such as acetylation, phosphorylation, etc

Figure 2. Three possible functions of PML NBs in relation to their liquid-like properties. Liquid properties are advantageous for the cells by providing the ability of fast and easy rearrangements of macromolecules. Yet, the separation of the ”liquid” nucleoplasm in several membrane-less condensates including PML NBs is essential to allow the formation of small reaction volumes with a different composition from the outside. Description of PML NBs as biomolecular condensates can illuminate the understanding of their function. We can envisage three important functions which may explain their roles in chromatin dynamics: (1) PML NBs may concentrate biochemical reactions. The biochemical environment within phase-separated PML NBs is different from the nucleoplasm and could serve to regulate (i) the kinetics of enzymatic reactions or (ii) the specificity of the modifications catalyzed. This is consistent with the described role of PML NBs as sumoylation hotspots, but could also apply for other modifications such as phosphorylation, acetylation, ubiquitination, or protein degradation. An example of the SUMOylation of a given client by UBC9 or of another client modification by a specific enzyme is shown. (2) PML NBs may buffer/sequester proteins via liquid-liquid phase separation of these client proteins. Increase in PML/client concentration may trigger accumulation of a given protein in PML NBs as a means to buffer the amount of the free protein in the nucleoplasm (as observed early for CBP for example). In addition, protein sequestration in PML NBs might affect their known activity as observed for DAXX. (3) PML NBs may help to organize specific nuclear domains, such as chromatin domains. PML NBs are interspersed in the active chromatin compartment and could potentially help to organize this compartment by pulling together genomic loci with similar transcriptional regulation. Of note, these three functions are not mutually exclusive and may serve altogether to regulate chromatin dynamics. Concentration of various factors in PML NBs together with specific genomic loci may help to catalyse specific reactions at given loci, as in the case of the ALT pathway for example (see Figure3).

modifications (e.g. SUMOylation) of the PML scaffold or of a client protein, modify the valency of free sites available, and thus the affinity between interacting modules regulat-ing PML NB composition (6). SUMOylation of PML is not required for PML NB formation but is essential for the re-cruitment of partners containing one or several SIM motifs (23–25,27,116,117). Indeed, DAXX possesses 1 SIM mo-tif, I733IVLSDSD740, at its C-terminus which is both

crit-ical for its localisation in PML NBs as shown by deletion experiments, and sufficient for the localization of a GFP-DAXXSIM fusion protein in PML NBs (27,118).

Interest-ingly, DAXX can also be SUMOylated (118–121), but the forced fusion of SUMO1 or SUMO2 with DAXX is in-sufficient to rescue the DAXXSIM localization in PML condensates. Hence, in normal cell conditions, the presence of a SIM with affinity for SUMOylated PML is necessary and sufficient for a constitutive localization of DAXX in PML NBs. SUMOylation of DAXX by UBC9 present in the PML NBs then enforces its sequestration within the condensates by intermolecular interactions with PML SIM (27).

Interestingly, bioinformatics analysis of chromatin

asso-ciated factors such as DEK or SETDB1, which can local-ize in PML NBs, identified putative SIM in these proteins with SETDB1’s SIM being crucial for its interaction with SUMOylated proteins (101,122,123). It is tempting to spec-ulate that these SIM motifs could be implicated in their recruitment to PML NBs, yet further studies will need to confirm the exact sequence requirements. Overexpression of the PML protein itself or increase in PML SUMOy-lation (eg following IFN treatment (38–40)) increases the number of available SUMO groups on PML (multivalency), which can trigger the switch-like recruitment of client pro-teins such as CBP (6,124). On the contrary, ectopic overex-pression of client proteins, such as HIRA, UBN1 or CBP, leads to their recruitment to PML NBs, which may result from an increased valency of the client upon higher con-centration and thus suggests a buffering mechanism for ex-cess nucleoplasmic protein (95,124,125) (as represented in Figure 2 item (2)). Thus, caution should be taken when concluding on the localization of a given protein only based on the overexpression of an ectopic form. SETDB1 as well as HIRA also possess putative SUMO sites and have experimentally been found in screens for

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Table 2. List of histone chaperones, histone modifiers or histone readers localizing within PML NBs. Only proteins with known localization in PML NBs are listed, those that interact with PML, but whose localization in PML NBs has not yet been proven, have been omitted. Presence of validated SUMOylation sites or SIM motifs is indicated, putative sites/motifs identified by bioinformatic analysis or in SUMO screens are not shown. Positions refers to human proteins unless stated otherwise. While HP1 has been shown to be SUMOylated (236), it remains to be determined whether this SUMOylation controls its localization in PML NBs. The function related to the localization in PML NBs is also depicted. n.d. : non determined. hMSCs : human mesenchymal stem cells. MARs : Matrix attachment regions

Protein Protein function SUMO SIM Recruitment

Function related to localization in PML NBs References for PML NB localization References for SUMO/SIM ATRX H3.3 histone chaperone n.d. n.d. Constitutive, DAXX-dependent Heterochromatin establishment (82–83) -CBP Histone acetyltransferase

n.d. n.d. Constitutive Transcriptional regulation via p53 acetylation (53,72–73) -DAXX H3.3 histone chaperone Multiple lysine residues

SIM1 IIVL (aa 7-10) and SIM2 IIVLSDSD (aa 733–740)

Constitutive Transcriptional regulation, heterochromatin establishment, H3.3 recruitment in PML NBs, H3.3-dependent chromatin assembly (25,61) (118) DEK H3.3 histone chaperone n.d. AKRE (aa 260–263) (not validated by mutation) Constitutive (hMSCs) Maintenance of an H3.3 soluble pool available for recruitment in PML NBs

(101) (101)

H3.3 Histone H3 variant found in transcriptionally active regions and specific heterochromatic regions n.d. n.d. Constitutive as well in senescence, DAXX-dependent

H3.3 soluble pool available for triage between histone chaperones

(84–85)

-HDAC7 Class IIA histone deacetylase n.d. n.d. Constitutive in a subset of PML NBs, increased upon TNF-␣ Transcriptional regulation (sequestration in PML NBs to relieve gene repression) (108) -HIRA complex H3.3 histone chaperone complex composed of HIRA, UBN1, CABIN1 and transiently ASF1A n.d. n.d. Stress-induced (senescence, IFN, viral infection) H3.3-dependent chromatin assembly in transcriptionally active regions, sequestration mechanism ? (78,95–100) -HP1 Heterochromatin protein 1 K84 + alternative usage of various lysines residues n.d. Constitutive as well as in senescence Heterochromatin establishment, in particular at cell-cycle genes during senescence

(75–78) (236) MOZ (KAT6A) Histone acetyltransferase n.d. n.d. Stress-induced (DNA damage, senescence) Transcriptional regulation via p53 acetylation (112)

-SATB1 Chromatin organizer by anchoring of MARs to the nuclear matrix, transcriptional regulator K744 n.d. Constitutive in a subset of PML NBs, SUMO-dependent Transcriptional regulation in immune cells, regulation of SATB1 levels by caspase-induced cleavage

(143,182) (182)

SETDB1 Histone H3K9 trimethyltransferase

n.d. IIEI (aa 125–129) Constitutive H3K9me3 heterochromatin establishment at specific loci (such as Id2 gene) + maintenance of PML NBs (102) (122) SIRT1 NAD-dependent histone deacetylase n.d. n.d. Stress-induced (PML-IV overexpression, senescence) Deacetylation of p53 leading to repression of p53-mediated transactivation (109) -TET2 Oxidation of 5mC to promote DNA demethylation n.d. n.d. Chemotherapy-induced, dependent on PML C-Terminus Chemotherapy-induced demethylation of specific genes (113) -TIP60 (KAT5) Histone acetyltransferase K430 and K451 n.d. UV-induced, SUMO-dependent, PML3-dependent

UV-induced DNA damage response (p53 recruitment in PML NBs and stabilization), SUMOylation promotes HAT activity, regulation of KAT5A stability

(110–111) (110)

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lated proteins (119–121,126). Whether these sites are essen-tial to enforce their localization in PML NBs or regulate their turnover as observed for Sp100 (115) remains to be tested.

Changes in affinity may also be regulated by post-translational modifications of the scaffold (PML) or of the client proteins. Phosphorylation adjacent to the SIM mo-tifs, as observed for the phosphoSIMs of PML and DAXX, leads to an increased affinity towards SUMO1 via interac-tion with specific SUMO1 lysine residues (127–130). On the contrary, acetylation of SUMO1 decreases the affinity for SIM, as observed for DAXX which then looses its localiza-tion in PML NBs (131), and thus participates in the regu-lation of client partitioning into biomolecular condensates. Of note, acetylation of SUMO1 at key lysine residues al-ters binding to the phosphoSIMs of PML or DAXX show-ing the structural plasticity of SUMO-SIM interactions that can be controlled by residue-specific post-translational modifications (132). Phosphorylation of HIRA by glycogen synthase kinase 3␤ (GSK-3␤) has been proposed to regu-late its localization in PML NBs upon senescence (133), but does not seem to play a role in IFN-mediated relocalization of HIRA in PML NBs (100). Recently, a large RNAi screen also identified Homeodomain-Interacting Protein Kinases (HIPK1 & 2) as important regulators of PML NBs compo-sition. Overexpression of these proteins led to a decreased accumulation of Sp100 in PML NBs, but not of PML it-self. This suggests a role of HIPK1 & 2 in controlling the condensation of proteins in PML NBs by phosphorylation (134). The use of kinase-dead enzymes should rule out a possible titration effect where HIPK1 & 2 overexpression could saturate SUMO sites via binding of their SIM (135), hence displacing Sp100.

Thus, PML NBs contain multiple chromatin-associated factors whose localization is regulated by a switch-like par-tionning between the diffuse and the condensed phase con-trolled by the multivalency of PML and of the client protein itself. We will now discuss the possible physical contacts of PML NBs with chromatin.

A physical connection of PML NBs with chromatin

The use of an analytic electron microscopic method called Electron Spectroscopic Imaging (ESI) was instrumental in precisely determining the nucleic acid-based regions and protein-based regions within and around PML NBs (14). Boisvert and colleagues demonstrated that the PML NB core is a protein-based structure and that PML NBs are devoid of nucleic acids in normal conditions. Yet, nascent RNA, as well as highly acetylated blocks of chromatin were found to accumulate in the vicinity of PML NBs sug-gesting an association of these nuclear bodies with tran-scriptionally active chromatin (14) (see below). Further ESI studies demonstrated that the protein cores of PML NBs are surrounded by chromatin fibers and make direct phys-ical contact with them, allowing the positional stability of PML NBs (58). PML NBs are also found adjacent to replication foci labelled by BrdU in middle-late S-phase cells (57).

While PML NBs physically contact chromatin, addi-tional studies explored their associations with specific

re-gions of the genome. The use of immuno-DNA FISH to combine immunolocalization of PML NBs with localiza-tion of specific genomic loci provided convincing data to demonstrate the specific association of PML NBs with cel-lular chromosomal loci. Using this approach, Shiels et al. demonstrated for the first time a non-random association of a specific locus, the major histocompatibility complex (MHC) on chromosome 6, with PML NBs in human pri-mary fibroblasts (136), which was consistent with the role of PML in the upregulation of MHC specific genes (137). The association of this gene-rich locus with PML NBs was neither dependent on transcription nor on cell cycle phase, and could be observed when the locus was placed on chro-mosome 18. Further immuno-DNA FISH studies showed a specific association of PML NBs with the TP53 gene lo-cus, but not BCL2 in jurkat cells (138) as well as a more general association of PML NBs with regions of high tran-scriptional activity (52). Of note, the association of PML NBs with specific loci might be cell-cycle specific since asso-ciation of the histone gene cluster was increased in S-phase when canonical histones genes are transcribed (52). Simi-larly, it was found that PML NBs are preferentially juxta-posed to centromeres during G2-phase (77). Juxtaposition of genomic loci to PML NBs may be a means to regulate specific gene expression (see below). PML NBs show sig-nificant association with the Oct3/4 locus in ESCs, with a decrease upon differentiation in Neural Precursor Cells (NPCs), correlating with the decrease in Oct3/4 expression (139). In addition, Salsman et al. showed that PML NBs are not only juxtaposed to the DDTI4 gene locus, but they are also closely associated with the DDTI4 RNA transcrip-tional foci, as shown by immuno-RNA FISH, and consis-tent with the decreased expression of DDIT4 upon PML loss (140).

Interestingly, IFN␥ increases the spatial proximity be-tween PML NBs and the MHC class II gene cluster and PML is required for the IFN␥-induced MHC class II gene transcription (141). In particular, the association of a gene from this locus, the DRA gene, with PML NBs is main-tained after IFN␥ shut-off and is required to keep a pro-longed permissive chromatin state on the DRA promoter (142). This underlines the importance of the PML NBs spatial proximity with specific loci to mediate epigenetic memory of a stimulus through cell divisions to increase re-sponsiveness of gene expression to future activation signals (142). The connection of PML with the MHC locus was fur-ther substantiated by genomic studies using ChIP showing that PML is directly associated with specific regions within the MHC class-I locus (143). Together with special AT-rich sequence binding protein 1 (SATB1), PML is involved in the chromatin-loop organization of the MHC class-I lo-cus and regulates a distinct set of genes within this lolo-cus upon IFN␥ treatment (143). Recent ChiP-Seq analysis of PML binding regions in MEFs also found PML enriched at heterochromatin gene-poor loci called PML-associated domains (PADs) (144). However, even if ChIP experiments overcome the a priori assumptions for selecting a genomic locus, ChIP cannot distinguish between the nucleoplasmic pool of PML and PML that is located within PML NBs. In particular, the recent ChIP-Seq analysis against PML in MEFs illustrates that most of the loci immunoprecipitated

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with PML do not localize in PML NBs and PML associa-tion at these loci is required to preserve their H3K9me3 het-erochromatic state (144). Immuno-FISH therefore remains an indispensable control to assess whether the association of PML with specific chromatin loci happens through PML NBs.

To overcome limitations of immuno-FISH and ChIP, the Bazett-Jones’ team developped a method called immuno-TRAP which allows the deposition of biotin onto DNA in close proximity to PML NBs. DNA can then be purified with streptavidin agarose beads and analyzed in a unbiased manner (145). Using various FOSMIDS, the authors con-firmed an interaction of PML NBs with TP53 locus and un-cover an association with the PML locus itself. The associa-tions observed were cell-type specific and dependent on the cell’s physiological state since IFN␣ treatment modified the loci association with PML NBs (145). Importantly, the use of an engineered APEX2 peroxidase fused to PML in mouse ESCs to mediate chromatin labeling and purification com-bined with deep-sequencing (ALaP-Seq) allowed the identi-fication of chromatin regions proximal to PML NBs (114). The authors confirmed the association of PML NBs with regulatory regions of active genes in a genome-wide man-ner, as well as identified novel hotspots regions, such as the short arm of the Y chromosome, frequently associated with PML NBs (114). The use of a PML RING domain mutant, that is diffuse in the nucleoplasm, only gave very few peaks demonstrating that the majority of the ALaP-Seq peaks tru-ely reflect chromatin interactions with PML NBs, but not with the nucleoplasmic pool of PML.

In parallel to the connection of PML NBs with cellular loci, it was early demonstrated that the genomes of specific viruses (HSV-1, Simian Virus 40 (SV40) and adenovirus) were also juxtaposed to PML NBs during the early stages of lytic virus infection (146,147). A role of PML NBs as potential docking sites for viruses favoring their replica-tion and/or transcription was then confirmed for HSV-1 (148,149), human papilloma virus 11 (150), Epstein-Barr Virus (151), and bovine papillomavirus (152), suggesting that PML NBs could facilitate the infectious process un-der certain circumstances. In contrast, the presence of a foreign plasmid transgene or of latent human immunode-ficiency virus (HIV) proviruses next to PML NBs was as-sociated with transcriptional silencing (107,153). These ex-amples suggest that cells could handle foreign genomes of viral origin in a non-random fashion, and that PML NBs are likely to show a Janus activity depending on the virus and the stage of the viral infection (see below).

PML NBs can contain DNA/chromatin in specific cases Specific cellular or viral loci can be juxtaposed to PML NBs. However, in certain cases, particular DNAs are lo-cated inside the nuclear bodies. The first example comes from telomerase-negative tumors and tumor-derived hu-man cell lines that have been shown to maintain their telom-eres length by a mechanism called alternative lengthening of telomeres (ALT) (154–156). ALT cells and tumors con-tain specific structures of PML NBs referred to as ALT-associated PML nuclear Bodies (APBs). High resolution microscopy images show that these APBs contain telomeric

DNA in the interior of the structure, in addition to the PML protein and its partners (16,157,158) (Figure3). In another specific pathology, the Immunodeficiency, Centromeric re-gion instability, and Facial anomalies syndrome (ICF), en-larged ring-like PML structures, namely giant PML NBs, were observed in ICF G2 nuclei (159). ICF is a rare auto-somal recessive disorder associated with mutations in the DNA-methyltransferase DNMT3B gene causing the hy-pomethylation and decondensation of the heterochromatic structure of satellite DNA mostly in pericentromeric re-gions of chromosomes 1 (1qh), 16 (16qh) and 9 (9qh) (160– 162). Giant PML NBs contain the undercondensed 1qh or 16qh heterochromatin in the inner core with PML form-ing the outer shell (159) (Figure3). Other PML NBs con-stituents, such as HP1, DAXX, ATRX, SP100, SUMO1, CBP and the DNA repair-associated factors BLM, TOP3A and BRCA1 were also found inside the structure adopting a specific multilayered organization (159).

In the case of viral infection with HSV-1, a dsDNA virus, the latent viral genome does not integrate in the host genome, and remains as a chromatinized episomal form in the nucleus of infected cells. We and others have shown by confocal microscopy that positioning of the latent HSV-1 genomes is not random and instead, the viral genome is en-cased in PML NBs forming structures called viral DNA-containing PML NBs (vDCP-NBs) or ND10-like (71,163– 165). vDCP NBs contain, just like APBs and giant PML NBs, most of the PML partners, including the DAXX– ATRX complex, as well as all members of the H3.3 histone chaperone HIRA complex (98–100) (Figure 3). Interest-ingly, a physical and functional association of the genome of an RNA virus, the hepatitis delta virus was observed with PML NBs. The particular antigenomic RNA co-localizes with PML NBs but contrarily to the APBs, giant PML NBs and vDCP NBs, resides at the edge of a rim-like structure that shows in the inside the presence of the PML, SP100, DAXX and SUMO-1 proteins (166). This peculiar associ-ation plays a role in viral RNA synthesis mediated by host RNA polymerase II (167), but has not been studied further and remains so far the only example of a viral RNA prod-uct closely associated with PML NBs. Finally, an exoge-nous cytomegalovirus promoter-containing transgene array is found at the center of PML NBs, with PML and SUMOs forming a ring structure around it, as observed by confocal microscopy, supporting the evidence of DNA in the inte-rior of PML NBs (168,169). Altogether these specific exam-ples show that PML NBs have a strong physical connection with specific genomic loci and can entrap particular DNAs, supporting an important role in regulating DNA-metabolic processes (see below).

Regulation of the physical connection of PML NBs with chro-matin loci

PML NBs are very dynamic entities whose number and size varies depending on the cell cycle and on various stimuli (61,170). During interphase, nucleoplasm is separated from the cytoplasm by the nuclear envelope forming a selective barrier. PML NBs exhibit apparent stability in the nucleus of unperturbed healthy cells. Yet they are actively remodeled during S-phase due to chromatin topological changes

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Figure 3. PML NBs directly regulate chromatin dynamics of DNA sequences found in the condensate. (A) Viral DNA-containing PML NBs (vDCP NBs) are specific PML NBs encasing the HSV-1 latent viral genome. Both H3.3 histone chaperone complexes (DAXX-ATRX and HIRA complexes) are found in these structures together with H3.3–H4. These complexes are essential for the H3.3 chromatinization of the virus, together with PML. H3.3 is decorated with the heterochromatin mark H3K9me3, which could be deposited by SETDB1 (question mark), a known client protein localizing constitutively in PML NBs. (B) PML NBs containing satellite DNA are found in the ICF syndrome in the form of giant PML NBs. These structures contain proteins organized in ordered concentric layers around the satellite DNA core, in the following order from the center: HP1 proteins, DAXX–ATRX complex, CBP/BLM/TOP3A, surrounded by a sphere of SUMO1/SP100 and then PML protein (concentric layers not shown). While the heterochromatin nature of the satellite DNA is atypical with absence of the constitutive H3K9me3 mark despite HP1 presence, the presence of␥H2A.X in some giant PML NBs (25%) nevertheless suggests that satellite DNA is associated with chromatin inside PML NBs. Of note, normal PML NBs can also contain satellite DNA in G2 phase. PML NBs-containing satellite DNA may help remodelling and maintenance of the heterochromatin structure present at late-replicating satellite DNA. (C) ALT-associated PML NBs (APBs) are a hallmark of the ALT pathway. Here we only focus on the chromatin dynamics in APBs, and neither display the numerous repair factors present in APBs nor the mechanisms involved in ALT. Telomeric DNA localizes within PML NBs together with specific chromatin-related factors such as SETDB1, ASF1, or HIRA. Recent data suggest that telomeric DNA repeats are more compact, with higher levels of H3K9me3 deposited by SETDB1 (i), and bound less TRF2 in ABPs than regular telomeres (ii), which would cause telomeric deprotection and promote telomeric recombination. Increase in TERRA transcription (orange lines) is also observed (iii) and fuels the ALT process by increasing RNA:DNA hybrids (iv) and thus replicative stress. Depletion of the histone chaperone Asf1 promotes histone management dysfunction during telomeric replication and is sufficient to trigger ALT (v).

out major changes in PML protein levels or biochemical al-terations. In S-phase, chromatin that undergoes replication retracts from PML NBs and actively pulls the PML NBs apart causing their fragmentation in smaller PML NBs by a fission mechanism (171). High rates of PML protein ex-change between the nucleoplasmic pool and the PML NBs then ensures that the nascent PML microbodies increase in size by G2. Of note, fusion events can also contribute to the regulation of PML NBs size in S-phase (171).

Reduction of the physical contacts between PML NBs and chromatin can also be induced by specific stresses, such as heat shock, transcriptional repression, apoptosis induc-tion, DNA damage or oxidative stress (58,114,172,173). This triggers the formation of newly formed microbodies by fission as well as in their increased mobility underscor-ing the importance of chromatin interactions for the struc-tural and morphological integrity as well as the dynamics of PML NBs (58,174). Increase in PML NBs number may also be linked to chromatin decondensation mediated by an ATM-KAP1 axis during DNA damage, or as observed upon HDAC inhibition (172,173). Biomolecular conden-sates can fuse, coalesce and drip, which are typical prop-erties of liquid assemblies (4). Fission and fusion events of

PML NBs observed across the cell cycle or following vari-ous stresses thus appear as a convincing feature that would sustain the hypothesis of a liquid-like behavior for these nu-clear bodies. Changes in the amount of PML NBs contacts with chromatin across the cell-cycle or following various stresses can thus provide many regulation opportunities for the cells that will need to be explored further.

PML NBs ARE IMPORTANT FOR THE CHROMA-TINIZATION OF VIRAL GENOMES

The discovery of the association of PML NBs with the genomes of several viruses suggests that PML NBs and their chromatin-related factors mediate their antiviral ac-tivity partly through this physical interaction. Remarkably, viruses have evolved several strategies to counteract these antiviral effects by encoding specific anti-PML NBs viral proteins. This is the case for HSV-1 infected cell protein 0 (ICP0), which induces the proteasomal-dependent degra-dation of SUMOylated forms of PML, leading to PML NBs disappearance. Other viruses directly target the SUMO modification of PML (by preventing it or removing it), thus altering the multivalent potential of PML and the

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tion of their protein composition by phase separation (for reviews (175–177).

The packaging of viral DNA with cellular histones car-rying specific post-translational modifications allows for a transcriptional control of viral expression (for review (178)). As mentioned above, latent HIV provirus juxta-posed to PML NBs is transcriptionally silent, while reac-tivation of the virus correlates with displacement of the provirus away from the nuclear bodies (107). Interestingly, the transcriptional repression activity of PML requires its binding to the latent chromatinized provirus, which al-lows the recruitment of the G9a methyltransferase respon-sible for H3K9 dimethylation (H3K9me2) on the provirus promoter. Knockdown of PML results in a decrease of provirus-bound G9a, a loss of H3K9me3 heterochromatin marks on the silent viral promoter and a concomitant gain in the transcriptionally-prone H3K4me3 marks (107). An-other compelling example of the involvement of PML NBs in the chromatinization of a viral genome came from our lab with regards to the chromatinization of the nucleosome-free HSV-1 genome entering the nucleus of the infected cells, prior to the establishment of latency (99). PML NBs en-case the latent viral genome together with the H3.3 chaper-one complexes DAXX-ATRX and HIRA, thus allowing the concentration of histone chaperones together with the viral genome in a condensed phase (99) (Figures2and3A). The two H3.3 histone chaperone complexes likely play redun-dant role in the chromatinization of latent HSV-1 genomes with H3.3-containing nucleosomes, together with the PML protein itself (99). Interestingly, H3.3 is modified with the repressive heterochromatin mark H3K9me3 on the HSV-1 latent genome and destabilization of PML NBs contain-ing the viral genome by ICP0 results in the recovery of a lytic transcriptional program (99), underscoring the repres-sive function of the PML NB-H3.3 axis in virus latency (Figure3A). However, HIRA mediated-deposition of H3.3 on HSV-1 genomes during the lytic phase is required for viral RNAs transcription (179), consistent with the dou-ble face of H3.3 which can thus be deposited in actively transcribed regions depending on the infectious context. Of note, the switch-like partitioning of HIRA in the PML-NBs following infection by HSV-1 (98–100), correlates with the transcriptional upregulation of host genes, including inter-feron stimulated genes (ISGs), and contributes to the in-trinsic and innate immune defenses against HSV-1 infection (98,100). Therefore, the PML NBs antiviral activity could act both directly through the chromatinization-associated transcriptional regulation of the viral genomes, and indi-rectly through the regulation of ISGs. PML NBs thus play an essential role in the regulation of viral chromatinization with specific histones variants/marks and are essential for the epigenetic control of viral expression.

PML NBs PARTICIPATE IN THE REGULATION OF CELLULAR CHROMATIN DYNAMICS

PML NBs and the dynamics of chromatin during transcrip-tional regulation

PML NBs are present in regions of high transcriptional ac-tivity (14,52). The recent ALaP-Seq analysis of chromatin

regions proximal to PML NBs confirms that PML NBs as-sociate primarily with regulatory regions of active genes in a genome-wide manner (114). In addition to their role in the transcriptional regulation of viral genes, they also regulate transcription of cellular loci (for review (180)). While the PML protein itself can act as a transcriptional co-activator or co-repressor, we focus here specifically on the interplay between PML NBs and transcriptional regulation through the prism of chromatin dynamics and in light of their liquid-like properties (Figure4).

First, PML NBs can regulate transcriptional activity through specific modifications of transcriptional factors as examplified for p53 acetylation and phosphorylation in senescence (see below) (Figure 4Ai). PML NBs are also known SUMOylation hotspots through concentra-tion of the SUMO E2-conjugating enzyme UBC9 (12,27). SUMOylation could serve to regulate client activity as ob-served for TIP60-induced HAT activity upon UV dam-age (110) (Figure 4Ai). Poly-SUMOylation may initiate polyubiquitination by the SUMO-targeted Ubiquitin lig-ase (STUbl) RNF4 and subsequent proteasome-mediated degration, as observed for the degradation of the PML-RAR␣ fusion protein (116,181) (Figure 4Aii). SUMOy-lation may also trigger caspase-dependent degradation of proteins within PML NBs as observed for SATB1, im-pairing its role in chromatin loop organization and tran-scriptional regulation (143,182) (Figure 4Aiii). However, the abundance of other chromatin related factors, such as HIRA, does not change upon relocalization in PML NBs (98,100) and it remains to be investigated whether SUMOy-lation could serve to regulate histone chaperone activity.

Second, PML NBs can regulate transcription by modu-lating the availability of chromatin associated factors within PML NBs. E2Fs transcription factors may be sequestered by pRb within PML NBs upon senescence preventing E2F target genes transcription (see below) (Figure4Aiv). On the contrary, sequestration of the histone deacetylase HDAC7, a potent transcriptional repressor, in PML NBs could par-ticipate in upregulation of MEF2 target genes (108) (Fig-ure4Av). Dynamic changes in histone chaperone localiza-tion might also be a means to fine-tune gene expression. The H3.3 histone chaperone DAXX acts as a potent tran-scriptional repressor and is a well-studied PML NBs com-ponent. Sequestration of DAXX in PML NBs releases tran-scriptional repression on reporter genes or specific cellular genes, such as glucocorticoid receptor target genes, whereas disruption of DAXX localization in PML NBs by ICP0 or expression of SUMOylation-defective PML mutant fails to relieve DAXX-mediated transcriptional repression (183– 185) (Figure4Av). HIRA localizes in PML NBs upon spe-cific stresses, such as IFN treatment, without any global change in the amount of HIRA RNA or protein levels (98,100). This anticipates a drop of HIRA concentration in the rest of the nucleoplasm through a sequestration mech-anism in PML NBs. We can hypothesize that the depletion of HIRA from genomic loci could have a global impact on H3.3 dynamics at specific genes located at a distance from PML NBs, but this titration effect remains to be investigated (Figure4Avi).

Third, PML NBs could participate in establishing chro-matin domains that are either permissive or refractory to

(14)

Figure 4. Role of PML NBs in transcriptional regulation. PML NBs has a dual effect on gene expression both facilitating or repressing expression of specific genes. (A) PML NBs regulates transcription through specific modifications of transcription factors or by modulating the availability of transcription factors or chromatin-related factors. (i) Upon Ras-induced senescence entry, p53 localizes in PML NBs which promotes its phosphorylation on serine 15 (not shown) as well as its acetylation on lysine 382 by CBP or MOZ, which may be counteracted by SIRT1. These PML-dependent modifications are required for p53 transactivation activity. TIP60 SUMO-dependent relocalization in PML NBs upon UV damage may also participate in p53 recruitment and stabilization (dashed arrow), thus favoring its transactivation activity. Oxidative stress can also trigger PML-dependent p53 activation conveying the ROS response (237). (ii) PML NBs can regulate proteins levels by SUMO-dependent poly-ubiquitination by RNF4 and subsequent proteasome-mediated degradation as observed for PML-RAR␣, or (iii) by caspase degradation as observed for SATB1. (iv) In senescence, PML NBs concentrate Protein Phosphatase 1 alpha (PP1␣) together with Rb preventing its CDK-dependent phosphorylation and thus inhibiting E2F which remains sequestered in PML NBs and cannot activate cell-cycle promoting genes. (v) The DAXX histone chaperone brings new H3.3-H4 dimers within PML NBs but may then be sequestered preventing the transcriptional repression of its target genes such as Glucocorticoid receptor (GR) target genes. HDAC7 may also be sequestered to prevent repression of MEF2 target genes. (vi) The role for HIRA complex localization in PML NBs remains more enigmatic (question mark). (B) PML NBs could also participate in establishing chromatin domains that are either permissive or refractory to transcription. (i) Interaction between SATB1 and PML is essential to establish a specific chromatin-loop structure at the MHC class I locus and may serve to regulate transcriptional activity of genes within this locus. (ii) PML NBs can also provide a transcriptionally-permissive chromatin environment to neighboring loci (dashed green circle). In particular, binding to the short arm of the Y-chromosome (region YS300) to PML NBs allows anchoring of specific Y-linked gene promoters that are located away from this region (dashed line). PML NBs allow the maintenance of their transcriptional activity by excluding DNMT3A and preventing DNA methylation on these proximal promoters. Specific transcription factors or chromatin-related factors located in PML NBs (orange factor) could also contribute to gene expression in these chromatin domains. (iii) On the contrary, PML NBs may help to concentrate HP1 proteins on specific loci, possibly through phase separation of heterochromatin (dashed red circle), to promote repression of genes such as E2F target genes. SETDB1 may also participate in creating a repressing heterochromatin environment by depositing H3K9me3 on gene promoters such as for the Id2 gene. However, it remains to be determined whether these repressed loci are found in vicinity of PML NBs (question mark).

transcription (Figure4B). A recent paradigm shift in the field of transcriptional regulation has put forward a phase separation model for transcriptional control, in which multi-molecular assemblies would form by phase separa-tion bridging enhancers and promoters allowing gene acti-vation (186). As biomolecular condensates contacting spe-cific chromatin loci, PML NBs could participate in forming specific transcriptional conditions on genomic loci. Using a novel CasDrop technology, a Crispr–Cas9-based optoge-netic technology allowing local concentration of droplets at specific genomic loci, Shin et al. recently showed that

con-densates form preferentially in low-density chromatin re-gions (like PML NBs) and are able to mechanically pull to-gether targeted genomic loci (187). Although CasDrop is an artificial system with the tethering of specific proteins able to phase separate onto genomic loci, the mechanical pulling of distal genomic loci may indeed occur in vivo for PML NBs. In particular, at the MHC locus, PML NBs might regulate transcription of specific genes through the formation of SATB1-associated specific chromatin loops, bringing closer some distal genes in the locus (143) (Fig-ure4Bi). In addition, using ALaP-Seq Kurihara et al.

Figure

Figure 1. Structure of PML and organization of PML NBs. (A) Structure of the PML protein scaffold
Table 1. Summary of LLPS criteria that are matched or not by canonical PML NBs. In this table, we put forward the experimental evidence that sustains or not the involvement of LLPS in biogenesis of canonical PML NBs
Figure 2. Three possible functions of PML NBs in relation to their liquid-like properties
Table 2. List of histone chaperones, histone modifiers or histone readers localizing within PML NBs
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